Within-Host Diversity of SARS-CoV-2 in COVID-19 Patients With Variable Disease Severities

Abstract

Background: The ongoing pandemic of SARS-COV-2 has already infected more than eight million people worldwide. The majority of COVID-19 patients either are asymptomatic or have mild symptoms. Yet, about 15% of the cases experience severe complications and require intensive care. Factors determining disease severity are not yet fully characterized. Aim: Here, we investigated the within-host virus diversity in COVID-19 patients with different clinical manifestations. Methods: We compared SARS-COV-2 genetic diversity in 19 mild and 27 severe cases. Viral RNA was extracted from nasopharyngeal samples and sequenced using the Illumina MiSeq platform. This was followed by deep-sequencing analyses of SARS-CoV-2 genomes at both consensus and sub-consensus sequence levels. Results: Consensus sequences of all viruses were very similar, showing more than 99.8% sequence identity regardless of the disease severity. However, the sub-consensus analysis revealed significant differences in within-host diversity between mild and severe cases. Patients with severe symptoms exhibited a significantly (p-value 0.001) higher number of variants in coding and non-coding regions compared to mild cases. Analysis also revealed higher prevalence of some variants among severe cases. Most importantly, severe cases exhibited significantly higher within-host diversity (mean = 13) compared to mild cases (mean = 6). Further, higher within-host diversity was observed in patients above the age of 60 compared to the younger age group. Conclusion: These observations provided evidence that within-host diversity might play a role in the development of severe disease outcomes in COVID-19 patients; however, further investigations are required to elucidate this association.

Keywords: COVID-19 severity; SARS-CoV-2; nonsynonymous mutations; virus quasispecies; within-host diversity.

Figure 1

Classification of SARS-CoV-2 viruses. Viruses were classified based on the lineage nomenclature system proposed in Rambaut et al. (2020). Numbers inside the boxes represent the total number of samples within each lineage.

Figure 2

Genomic variations detected in SARS-CoV-2 viruses. (A) Genomic positions and frequencies of variants found in the SARS-CoV-2 genome (55-29640) of 46 patients. The y-axis represents the frequency of variants, and the x-axis represents the genomic position of variants. (B) A tree map presenting types of variants found in SARS-CoV-2 genomes of mild (green) and severe (red) cases. (C) Colored parts of the circles represent variants seen in more than one patient.

Figure 3

Phylogenetic tree of sequenced SARS-CoV-2 viruses (n = 46 samples). Maximum likelihood phylogeny was reconstructed under the general-time reversible model (GTR) as inferred by model testing. Nucleotide sequence alignment shows mutations found at the consensus level (>50% frequency) with respect to the reference strain (MN908947).

Figure 4

Comparison of variant prevalence in mild cases (green) and severe cases (red). Colored parts of the circles (on the left) represent variants seen in more than one patient. Only variants found in more than one patient were used to generate bar charts. Bar charts demonstrate the prevalence of each variant in patients within each group (severe vs. mild). Bars with darker colors demonstrate variants detected at the consensus sequence level. P-values are indicated as follows: ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05.

Figure 5

Variants in the SARS-CoV-2 spike glycoprotein. (A) Genomic position and frequency of variants detected in S genes of all viruses. (B) Schematic representation showing consensus mutations found in mild (green) and severe (red) cases. D614G mutation was found in both mild and severe cases.

Figure 6

Assessment of within-host SARS-CoV-2 diversity. (A) Total number of variants detected in each patient with mild (green) and severe (red) symptoms. (B) Number of synonymous (S) and non-synonymous (NS) mutations found within each patient of the two groups (mild vs. severe cases). P-values are indicated as follows: ****p < 0.0001, ***p < 0.001.

Figure 7

Correlation between within-host variation (number of variants within each patient) and age (A), severity score (B), and number of comorbidities (C). Patients with mild symptoms are indicated in green while patients with severe symptoms are indicated in red. Shaded circles in (B–E) indicate patients older than 50 years.